Organo-arsenoxide compounds and use thereof

ABSTRACT

The present invention relates to organo-arsenoxide compounds and to methods for their synthesis. The invention also relates to pharmaceutical compositions comprising these compounds and to their use in the treatment of diseases and disorders, in particular proliferative diseases and disorders, including treatment of solid tumors and leukaemia.

This application is a US national phase of International Application No.PCT/AU2007/001676 filed on Nov. 1, 2007, the disclosure of which isherein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to organo-arsenoxide compounds and tomethods for their synthesis. The invention also relates topharmaceutical compositions comprising these compounds and to their usein the treatment of diseases and disorders, including proliferativediseases and disorders.

BACKGROUND OF THE INVENTION

Arsenical compounds have been used in the past as therapeutic agents forthe treatment of disease. However, the inherent toxicities of arsenicalcompounds and their generally unfavourable therapeutic index haveessentially precluded their use as pharmaceutical agents.

Organo-arsenoxide compounds have been disclosed in WO 01/21628. Suchcompounds are described as having antiproliferative properties useful inthe therapy of proliferative diseases. WO 04/042079 discloses the use oforgano-arsenoxide compounds for inducing the mitochondrial permeabilitytransmission (MPT) and the use of organo-arsenoxide compounds forinducing apoptosis, particularly in endothelial cells. Theorgano-arsenoxide compounds described in WO 01/21628 and WO 04/042079have a substantially cell-membrane impermeable pendant group linked viaa linking group to an arsenoxide group. Neither WO 01/21628 nor WO04/042079 specifically disclose compounds of formula (I) according tothe present invention.

Patients with acute promyelocytic leukaemia (APL) can suffer relapsefollowing treatment with the current therapy, all-trans retinoic acid.In such cases, arsenic trioxide is considered the treatment of choice(Reiter et al., 2004). Arsenic trioxide is a trivalent arsenical thatselectively kills APL cells. Arsenic trioxide is also showing promisefor the treatment of myelodysplastic syndrome (Vey, 2004), a disease forwhich no standard treatment currently exists.

However, inorganic arsenicals, such as arsenic trioxide, have long beenrecognised as a poison and carcinogen when present in the body at levelsthat exceed its capacity to detoxify the metalloid and are associatedwith many adverse side effects.

There is a need for alternative therapies for treating proliferativediseases, such as cancer (including treatment of solid tumors), andrelated conditions. In particular, there is a need for alternativetherapies for treating APL, including acute myelocytic leukaemia (AML).There is also a need for a therapeutic treatment for myelodysplasticsyndrome.

The present invention relates to a group of arsenoxide compoundscomprising an optionally substituted amino acid residue linked via alinking group to a phenylarsenoxide group. Compounds according to thepresent invention may have one or more advantage(s) over known arsenicalcompounds, such as arsenic trioxide and the arsenoxide compoundsdisclosed in WO 01/21628 or WO 04/042079, including the compound4-(N—(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO), particularlywhen used for the treatment of proliferative disease, such as cancer(e.g., solid tumors).

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a compound of generalformula (I):

wherein

-   -   the As(OH)₂ group may be ortho-, meta- or para- to the N-atom on        the phenyl ring;    -   R¹ is selected from hydrogen and C₁₋₃ alkyl;    -   R² and R³ may be the same or different and are independently        selected from hydrogen, optionally substituted C₁₋₃ alkyl,        optionally substituted cyclopropyl, optionally substituted C₂₋₃        alkenyl; and optionally substituted C₁₋₃ alkoxy;    -   R⁴ and R⁵ may be the same or different and are independently        selected from hydrogen, optionally substituted C₁₋₃ alkyl,        optionally substituted cyclopropyl, optionally substituted C₂₋₃        alkenyl; and optionally substituted C₁₋₃ alkoxy;    -   m is an integer selected from 1, 2 and 3;    -   n is an integer selected from 1, 2 and 3;    -   * indicates a chiral carbon atom; and    -   salts and hydrates thereof.

In a second aspect the present invention relates to a pharmaceuticalcomposition comprising at least one compound of formula (I) according tothe first aspect of the invention, together with a pharmaceuticallyacceptable excipient, diluent or adjuvant.

In another aspect the present invention relates to a method of treatinga proliferative disease in a vertebrate, the method comprisingadministering to the vertebrate a therapeutically effective amount of acompound of formula (I) according to the first aspect of the invention,or a composition according to the second aspect of the invention. Theproliferative disease may be cancer, such as a solid tumour.

In a further aspect the present invention relates to a method ofinhibiting angiogenesis in a vertebrate, comprising administering to thevertebrate an effective amount of a compound of formula (I) according tothe first aspect of the invention, or a composition according to thesecond aspect of the invention.

In another aspect the present invention relates to a method of inducingthe Mitochondrial Permeability Transition (MPT) in a vertebratecomprising administering to the vertebrate a therapeutically effectiveamount a compound of formula (I) according to the first aspect of theinvention, or a composition according to the second aspect of theinvention.

In a further aspect the present invention relates to a method ofinducing apoptosis in proliferating mammalian cells, comprisingadministering to the mammal an apoptosis-inducing amount of a compoundof formula (I) according to the first aspect of the invention, or acomposition according to the second aspect of the invention.

In another aspect the invention relates to a method of treatingleukaemia or myelodysplastic syndrome in a vertebrate, comprisingadministering to the vertebrate a therapeutically effective amount of acompound of formula (I) according to the first aspect of the invention,or a composition according to the second aspect of the invention.

In a further aspect the present invention relates to the use of at leastone compound of formula (I) according to the first aspect of theinvention in the manufacture of a medicament for treating aproliferative disease in a vertebrate. The proliferative disease may becancer, such as a solid tumour.

In another aspect the present invention relates to the use of at leastone compound of formula (I) according to the first aspect of theinvention in the manufacture of a medicament for inhibiting angiogenesisin a vertebrate.

In yet another aspect the present invention relates to the use of atleast one compound of formula (I) according to the first aspect of theinvention in the manufacture of a medicament for inducing the MPT in avertebrate.

In a further aspect the present invention relates to the use of at leastone compound of formula (I) according to the first aspect of theinvention in the manufacture of a medicament for inducing apoptosis inproliferating mammalian cells.

In another aspect the present invention relates to the use of at leastone compound of formula (I) according to the first aspect of theinvention in the manufacture of a medicament for treating leukaemia in avertebrate.

DEFINITIONS

The following are some definitions that may be helpful in understandingthe description of the present invention. These are intended as generaldefinitions and should in no way limit the scope of the presentinvention to those terms alone, but are put forth for a betterunderstanding of the following description.

Unless the context requires otherwise or specifically stated to thecontrary, integers, steps, or elements of the invention recited hereinas singular integers, steps or elements clearly encompass both singularand plural forms of the recited integers, steps or elements.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers, but not the exclusionof any other step or element or integer or group of elements orintegers. Thus, in the context of this specification, the term“comprising” means “including principally, but not necessarily solely”.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

In the context of this specification, BRAO refers to4-(2-bromoacetylamino)-benzenearsonic acid; CAO refers to4-(N—(S-cysteinylacetyl)amino)-phenylarsinous acid; GSAO refers to4-(N—(S-glutathionylacetyl)amino)phenylarsinous acid; and PENAO refersto 4-(N—(S-penicillaminylacetyl)amino)phenylarsinous acid[“(S)-Penicillamine-arsenoxide”].

As used herein, the term “C₁₋₃ alkyl group” includes within its meaningmonovalent (“alkyl”) and divalent (“alkylene”) straight chain orbranched chain saturated aliphatic groups having from 1 to 3 carbonatoms. Thus, for example, the term C₁₋₃ alkyl includes methyl, ethyl,1-propyl, and isopropyl.

The term “C₂₋₃ alkenyl group” includes within its meaning monovalent(“alkenyl”) and divalent (“alkenylene”) straight or branched chainunsaturated aliphatic hydrocarbon groups having from 2 to 3 carbon atomsand at least one double bond anywhere in the chain. Unless indicatedotherwise, the stereochemistry about each double bond may beindependently cis or trans, or E or Z as appropriate. Examples ofalkenyl groups include ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl,and 2-propenyl.

The term “C₂₋₃ alkynyl group” as used herein includes within its meaningmonovalent (“alkynyl”) and divalent (“alkynylene”) unsaturated aliphatichydrocarbon groups having from 2 to 3 carbon atoms and having at leastone triple bond. Examples of alkynyl groups include but are not limitedto ethynyl, 1-propynyl.

The term “alkoxy” as used herein refers to straight chain or branchedalkyloxy (i.e, O-alkyl) groups, wherein alkyl is as defined above.Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, andisopropoxy.

The term “amino” as used herein refers to groups of the form—NR^(a)R^(b) wherein R^(a) and R^(b) are individually selected fromhydrogen, optionally substituted (C₁₋₄)alkyl, optionally substituted(C₂₋₄)alkenyl, optionally substituted (C₂₋₄)alkynyl, optionallysubstituted (C₆₋₁₀)aryl and optionally substituted aralkyl groups, suchas benzyl. The amino group may be a primary, secondary or tertiary aminogroup.

In the context of this specification the term “arsenoxide” is synonymouswith “arsinous acid” and refers to the moiety As(OH)₂, which may also berepresented as As═O.

The term “amino acid” as used herein includes naturally andnon-naturally occurring amino acids, as well as substituted variantsthereof. Thus, (L) and (D) forms of amino acids are included in thescope of the term “amino acid”. The term “amino acid” includes withinits scope glycine, alanine, valine, leucine, isoleucine, methionine,proline, phenylalanine, tryptophan, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,arginine, and histidine. The backbone of the amino acid residue may besubstituted with one or more groups independently selected from(C₁₋₆)alkyl, halogen, hydroxy, hydroxy(C₁₋₆)alkyl, aryl, e.g, phenyl,aryl(C₁₋₃)alkyl, e.g, benzyl, and (C₃₋₆)cycloalkyl.

The term “C₆₋₁₀ aryl” or variants such as “arylene” as used hereinrefers to monovalent (“aryl”) and divalent (“arylene”) single,polynuclear, conjugated and fused residues of aromatic hydrocarbonshaving from 6 to 10 carbon atoms. Examples of aromatic groups includephenyl, and naphthyl.

The term “arylalkyl” or variants such as “aralkyl” as used herein,includes within its meaning monovalent (“aryl”) and divalent(“arylene”), single, polynuclear, conjugated and fused aromatichydrocarbon radicals attached to divalent, saturated, straight orbranched chain alkylene radicals. Examples of arylalkyl groups includebenzyl.

The term “C₃₋₈ heterocycloalkyl” as used herein, includes within itsmeaning monovalent (“heterocycloalkyl”) and divalent(“heterocycloalkylene”), saturated, monocyclic, bicyclic, polycyclic orfused hydrocarbon radicals having from 3 to 8 ring atoms, wherein from 1to 5, or from 1 to 3, ring atoms are heteroatoms independently selectedfrom O, N, NH, or S. The heterocycloalkyl group may be C₃₋₆heterocycloalkyl. The heterocycloalkyl group may be C₃₋₅heterocycloalkyl. Examples of heterocycloalkyl groups includeaziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, quinuclidinyl,azetidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrofuranyl,tetrahydropyranyl, and the like.

The term “C₅₋₂₀ heteroaromatic group” and variants such as “heteroaryl”or “heteroarylene” as used herein, includes within its meaningmonovalent (“heteroaryl”) and divalent (“heteroarylene”), single,polynuclear, conjugated and fused aromatic radicals having from 5 to 20atoms, wherein 1 to 6 atoms, or 1 to 4, or 1 to 2 ring atoms areheteroatoms independently selected from O, N, NH and S. Theheteroaromatic group may be C₅₋₁₀ heteroaromatic. The heteroaromaticgroup may be C₅₋₈ heteroaromatic. Examples of heteroaromatic groupsinclude pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 2,2′-bipyridyl,phenanthrolinyl, quinolinyl, isoquinolinyl, imidazolinyl, thiazolinyl,pyrrolyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, isothiazolyl,triazolyl, and the like.

The term “halogen” or variants such as “halide” or “halo” as used hereinrefers to fluorine, chlorine, bromine and iodine.

The term “heteroatom” or variants such as “hetero-” as used hereinrefers to O, N, NH and S.

The term “optionally substituted” as used herein means the group towhich this term refers may be unsubstituted, or may be substituted withone or more groups independently selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, haloalkyl,haloalkynyl, hydroxyl, hydroxyalkyl, alkoxy, thioalkoxy, alkenyloxy,haloalkoxy, haloalkenyloxy, NO₂, NR^(a)R^(b), nitroalkyl, nitroalkenyl,nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine,alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy,alkylsulfonyloxy, heterocycloxy, heterocycloamino, haloheterocycloalkyl,alkylsulfenyl, alkylcarbonyloxy, alkylthio, acylthio,phosphorus-containing groups such as phosphono and phosphinyl, aryl,heteroaryl, alkylaryl, aralkyl, alkylheteroaryl, cyano, cyanate,isocyanate, CO₂H, CO₂alkyl, C(O)NH₂, —C(O)NH(alkyl), and —C(O)N(alkyl)₂.Preferred substituents include C₁₋₃ alkyl, C₁₋₃ alkoxy,—CH₂—(C₁₋₃)alkoxy, C₆₋₁₀ aryl, e.g., phenyl, —CH₂-phenyl, halo,hydroxyl, hydroxy(C₁₋₃)alkyl, and halo-(C₁₋₃)alkyl, e.g., CF₃, CH₂CF₃.Particularly preferred substituents include C₁₋₃ alkyl, C₁₋₃ alkoxy,halo, hydroxyl, hydroxy(C₁₋₃)alkyl, e.g., CH₂OH, and halo-(C₁₋₃)alkyl,e.g., CF₃, CH₂CF₃.

In the context of this specification the term “administering” andvariations of that term including “administer” and “administration”,includes contacting, applying, delivering or providing a compound orcomposition of the invention to an organism, or a surface by anyappropriate means.

In the context of this specification, the term “vertebrate” includeshumans and individuals of any species of social, economic or researchimportance including but not limited to members of the genus ovine,bovine, equine, porcine, feline, canine, primates (including human andnon-human primates), rodents, murine, caprine, leporine, and avian. In apreferred embodiment the vertebrate is a human.

In the context of this specification, the term “treatment”, refers toany and all uses which remedy a disease state or symptoms, prevent theestablishment of disease, or otherwise prevent, hinder, retard, orreverse the progression of disease or other undesirable symptoms in anyway whatsoever.

In the context of this specification the term “effective amount”includes within its meaning a sufficient but non-toxic amount of acompound or composition of the invention to provide a desired effect.Thus, the term “therapeutically effective amount” includes within itsmeaning a sufficient but non-toxic amount of a compound or compositionof the invention to provide the desired therapeutic effect. The exactamount required will vary from subject to subject depending on factorssuch as the species being treated, the sex, age and general condition ofthe subject, the severity of the condition being treated, the particularagent being administered, the mode of administration, and so forth.Thus, it is not possible to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” may bedetermined by one of ordinary skill in the art using only routineexperimentation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structure of (S)-Penicillamine-arsenoxide (“PENAO”).

FIG. 2. ¹H-NMR spectrum of (S)-Penicillamine-arsenoxide.

FIG. 3. 2D ¹H-¹³C multiple bond correlation data for(S)-Penicillamine-arsenoxide. Long-range coupling was observed betweenthe acyl hydrogens of 4-(2-bromoacetylamino)benzenearsonic acid (“BRAO”)(δ 3.55) and the penicillamine quaternary carbon (δ 46.75). The NMRspectrum was recorded in D₂O on a 300 MHz Bruker, dual channel probe NMRspectrometer.

FIG. 4. Mass Spectrometry: sodiated mass peak observed at 413.011678(δ1.5 ppm from calculated). Rapid alkyl ester formation occurs dependingon the alcohol solvent used e.g, if the sample is run in methanol, themain peaks observed are +15 or +30 mass units.

FIG. 5. (S)-Penicillamine-arsenoxide inhibits proliferation of BAE cellswith an IC₅₀ of 0.4 μM. The pentavalent arsenical compound(S)-Penicillamine-arsenonic acid, has no effect on proliferation. Thedata points are mean±SD of three experiments performed in triplicate.

FIG. 6. Comparison of the effects of (S)-Penicillamine-arsenoxide on BAEproliferation versus viability. The data points are mean±SD of threeexperiments performed in triplicate.

FIG. 7. (S)-Penicillamine-arsenoxide is as good an inhibitor of APL cellproliferation as arsenic trioxide. Number of viable NB4 cells remainingafter 72 h incubation with increasing concentrations of arsenictrioxide, Penicillamine-arsenoxide or GSAO. Data points are the mean±SDof triplicate determinations.

FIG. 8. (S)-Penicillamine-arsenoxide is more efficient than GSAO atinducing the mitochondrial permeability transition. Mitochondrialswelling induced by 100 μM GSAO or (S)-Penicillamine-arsenoxide asmeasured by decrease in light scattering at 520 nm over 30 min. Thetraces are representative of two experiments performed in duplicate ontwo different mitochondrial preparations.

FIG. 9. (S)-Penicillamine-arsenoxide accumulates in BAE cells at a˜70-fold faster rate than GSAO. BAE cells were incubated for up to 4 hin presence of 50 μM GSAO or (S)-Penicillamine-arsenoxide and cytosolicarsenic was measured by inductively coupled plasma spectrometry. Thedata points and error bars are the mean±SD of triplicate determinationsand is representative of two experiments.

FIG. 10. Inhibition of cell-surface OATP blunts cellular accumulation of(S)-Penicillamine-arsenoxide and anti-proliferative activity A.Inhibition of (S)-Penicillamine-arsenoxide accumulation in endothelialcells by the OATP inhibitor, DIDS. Cells were pretreated or not with 500μM DIDS for 30 min and then incubated with 20 μM(S)-Penicillamine-arsenoxide for 2 h. Arsenic content was determined byICPMS. Values are mean±SD of triplicate determinations. The results arerepresentative of two experiments. **: p<0.01. B. DIDS blunts GSAOanti-proliferative activity in endothelial cells. BAE cells werepretreated or not with 300 μM DIDS for 30 min and then incubated with1.5 μM (S)-Penicillamine-arsenoxide for 24 h. Cell viability wasdetermined using MTT. Results are expressed as percentage of control.Values are mean±SD of triplicate determinations. Results arerepresentative of two experiments. **: p<0.01.

FIG. 11. (S)-Penicillamine-arsenoxide is pumped out of cells by MRP1/2.A BAE cells were incubated for up to 2 h with 50 μM(S)-Penicillamine-arsenoxide in the absence or presence of 4H10 (5 μM)or MK-571 (25 μM) and cytosolic arsenic was measured by inductivelycoupled plasma spectrometry. The data points and error bars are themean±SD of quadruplicate determinations and is representative of twoexperiments. B Effect of the MRP1/2 inhibitors, 4H10 (2 μM) and MK-571(15 μM), on Penicillamine-arsenoxide (0.3 μM) inhibition of BAE cellproliferation. The MRP inhibitors were incubated for 30 min with thecells prior to incubation with (S)-Penicillamine-arsenoxide for 72 h.The data points and error bars are the mean±SD of triplicatedeterminations. *** is p<0.001, ** is p<0.01

FIG. 12. Depletion of cellular glutathione increases(S)-Penicillamine-arsenoxide anti-proliferative activity. BAE cells wereco-treated with (S)-Penicillamine-arsenoxide and the indicatedconcentrations of BSO for 72 h and the IC₅₀ for proliferation arrest wascalculated. The data points and error bars are the mean±SD from twoexperiments performed in triplicate.

FIG. 13. Inhibition of human pancreatic carcinoma tumour growth bycontinuous subcutaneous administration of (S)-Penicillamine-arsenoxide.BxPC-3 tumours were established in the proximal midline of female 7 to 9week old BalbC nude mice. Mice bearing ˜50 mm³ tumours were implantedwith 28 day alzet micro-osmotic pumps subcutaneously in the flank. Thepumps delivered 0.25, 0.5 or 1 mg/kg/day (S)-Penicillamine-arsenoxide in100 mM glycine (vehicle).

FIG. 14. Production of CAO by enzymatic cleavage of GSAO.

FIG. 15. HPLC analysis of GSAO and CAO. 5 nmoles of GSAO (part A) or CAO(part B) was resolved on a C18 reverse phase column and detected byabsorbance at 256 nm.

FIG. 16. CAO accumulates more rapidly in cells and have greateranti-proliferative activity than GSAO. A. CAO accumulates in cells at amuch faster rate than GSAO. BAE cells were incubated with 50 μM GSAO orCAO for 4 h. Cellular arsenic levels were determined by ICPMS. The ratesof accumulation are GSAO and CAO are 0.03 and 0.20 nmol As atoms per 10⁶cells, respectively. Data points are the mean±SD of threedeterminations. The results are representative of two experiments. B.CAO is exported from cells by the multidrug resistance associatedprotein 1. BAE cells pretreated for 30 min with 10 μM of the MRP-1inhibitor 4H10 were incubated with 50 μM GSAO or CAO for 2 h. Cellulararsenic levels were determined by ICPMS. Data points are the mean±SD ofthree determinations. The results are representative of two experiments.C. GSAO and CAO IC₅₀ values for proliferation arrest of endothelialcells. BAE cells were incubated with 0.8-100 μM GSAO or CAO for 24, 48or 72 h. Cell viability was determined using MTT. Results are expressedas percentage of control. Values are mean±SD of triplicatedeterminations. Results are representative of three experiments.

FIG. 17. CAO triggers the mitochondrial permeability transition.Mitochondria were incubated with nil (●), 150 μM Ca²⁺ and 6 mM Pi (∘) or200 μM CAO (▴) and swelling monitored by decrease in light scattering at520 nm over 60 min. The traces are representative of two experiments.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention relates to organo-arsenoxide compounds comprisingan optionally substituted amino acid moiety linked via a linker group toa phenylarsenoxide group.

Organo-arsenoxide compounds in accordance with the present inventionhave a substituted or unsubstituted amino acid moiety. Examples of aminoacid moieties include cysteinyl, substituted cysteinyl, for examplepenicillaminyl (also known as β,β-dimethylcysteinyl or3-mercaptovalinyl), optionally substituted alaninyl, optionallysubstituted mercaptoalaninyl, optionally substituted valinyl, optionallysubstituted 4-mercaptovalinyl, optionally substituted leucinyl,optionally substituted 3- or 4-, or 5-mercaptoleucinyl, optionallysubstituted isoleucinyl, or optionally substituted 3-, 4- or5-isoleucinyl. In a preferred embodiment of the invention the amino acidmoiety is β,β-dimethylcysteinyl (“penicillaminyl”). In anotherembodiment of the invention the amino acid moiety is (S)-penicillaminyl.In another embodiment of the invention the amino acid moiety iscysteinyl. The amino acid moiety may have (L), (D), (R) or (S)configuration. Optional substituents include C₁₋₃ alkyl, cyclopropyl,C₁₋₃ alkoxy, —CH₂—(C₁₋₃)alkoxy, C₆₋₁₀ aryl, —CH₂-phenyl, halo, hydroxyl,hydroxy(C₁₋₃)alkyl, and halo-(C₁₋₃)alkyl, e.g., CF₃, CH₂CF₃. Inpreferred embodiments the optional substituents are independentlyselected from hydroxyl, methoxy, halo, methyl, ethyl, propyl,cyclopropyl, CH₂OH and CF₃.

The linker group of the organoarsenoxide compounds in accordance withthe present invention is a substituted or unsubstituted acetamido group.In one embodiment the linker group is an unsubstituted acetamido group.

In particular, the invention relates to compounds of general formula(I):

wherein

-   -   the As(OH)₂ group may be ortho-, meta- or para- to the N-atom on        the phenyl ring;    -   R¹ is selected from hydrogen and C₁₋₃ alkyl;    -   R² and R³ may be the same or different and are independently        selected from hydrogen, optionally substituted C₁₋₃ alkyl,        optionally substituted cyclopropyl, optionally substituted C₂₋₃        alkenyl; and optionally substituted C₁₋₃ alkoxy;    -   R⁴ and R⁵ may be the same or different and are independently        selected from hydrogen, optionally substituted C₁₋₃ alkyl,        optionally substituted cyclopropyl, optionally substituted C₂₋₃        alkenyl; and optionally substituted C₁₋₃ alkoxy;    -   m is an integer selected from 1, 2 and 3;    -   n is an integer selected from 1, 2 and 3;    -   * indicates a chiral carbon atom; and    -   salts and hydrates thereof.

Preferred embodiments of the compounds of general formula (I) aredescribed below. It should be understood that any one or more of theembodiment(s) disclosed herein may be combined with any otherembodiment(s), including preferred embodiment(s).

Optional substituents may be the same or different and are independentlyselected from C₁₋₃ alkyl, cyclopropyl, C₁₋₃ alkoxy, —CH₂—(C₁₋₃)alkoxy,C₆₋₁₀ aryl, —CH₂-phenyl, halo, hydroxyl, hydroxy(C₁₋₃)alkyl, andhalo-(C₁₋₃)alkyl, e.g, CF₃, CH₂CF₃. In one embodiment the optionalsubstituents are independently selected from hydroxyl, methoxy, halo,methyl, ethyl, propyl, cyclopropyl, CH₂OH and CF₃. In one embodimentthere are no optional substituents.

The As(OH)₂ group may be ortho- or para- to the N-atom on the phenylring. In one embodiment, the As(OH)₂ group is para- to the N-atom on thephenyl ring. In another embodiment the As(OH)₂ group is ortho- to theN-atom on the phenyl ring.

R¹ may be hydrogen, methyl or ethyl. In one embodiment R¹ is hydrogen.

R² and R³ may be the same or different. R² and R³ may be independentlyselected from hydrogen, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₁₋₃ alkoxy,halo(C₁₋₃)alkoxy, hydroxy(C₁₋₃)alkyl and halo(C₁₋₃)alkyl. In a preferredembodiment R² and R³ may be independently selected from hydrogen,methyl, ethyl, methoxy, vinyl, CH₂OH, CF₃ and OCF₃. In another preferredembodiment R² and R³ may be independently selected from hydrogen, methyland ethyl. In another embodiment R² is methyl and R³ is hydrogen. Inanother embodiment R² and R³ are both hydrogen.

R⁴ and R⁵ may be the same or different. R⁴ and R⁵ may be independentlyselected from hydrogen, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₁₋₃ alkoxy,halo(C₁₋₃)alkoxy, hydroxy(C₁₋₃)alkyl and halo-(C₁₋₃)alkyl. In apreferred embodiment R⁴ and R⁵ may be independently selected fromhydrogen, methyl, ethyl, methoxy, vinyl, hydroxy(C₁₋₃)alkyl, CF₃ andOCF₃. In another preferred embodiment R⁴ and R⁵ may be independentlyselected from hydrogen, methyl, ethyl and CH₂OH. In another embodimentR⁴ is methyl or ethyl and R⁵ is hydrogen or methyl. In anotherembodiment R⁴ is methyl and R⁵ is hydrogen. In another embodiment R⁴ andR⁵ are both hydrogen. In another embodiment R⁴ and R⁵ are both methyl.

In one embodiment m is 1 or 2. In another embodiment n is 1 or 2. Inanother embodiment m and n are both 1.

In one embodiment of compounds of formula (I), the As(OH)₂ group isortho- or para- to the N-atom on the phenyl ring; R¹ is hydrogen ormethyl; R² and R³ are independently selected from hydrogen, C₁₋₃ alkyl,C₂₋₃ alkenyl, C₁₋₃ alkoxy, halo-(C₁₋₃)alkoxy, hydroxy(C₁₋₃)alkyl andhalo(C₁₋₃)alkyl; R⁴ and R⁵ are independently selected from hydrogen,C₁₋₃ alkyl, C₂₋₃ alkenyl, C₁₋₃ alkoxy, halo(C₁₋₃)alkoxy,hydroxy(C₁₋₃)alkyl and halo(C₁₋₃)alkyl; m is 1 or 2; and n is 1 or 2.

In another embodiment of compounds of formula (I), the As(OH)₂ group isortho- or para- to the N-atom on the phenyl ring; R¹ is hydrogen ormethyl; R² and R³ are independently selected from hydrogen, methyl,ethyl, methoxy, vinyl, CH₂OH, CF₃ and OCF₃; R⁴ and R⁵ are independentlyselected from hydrogen, methyl, ethyl, CH₂OH, methoxy, vinyl, CF₃ andOCF₃; m is 1; and n is 1.

In a further embodiment of compounds of formula (I), the As(OH)₂ groupis ortho- or para- to the N-atom on the phenyl ring; R¹ is hydrogen ormethyl; R² and R³ are independently selected from hydrogen, methyl andethyl; R⁴ and R⁵ are independently selected from hydrogen, methyl andethyl; m is 1; and n is 1.

In another embodiment of compounds of formula (I), the As(OH)₂ group isortho- or para- to the N-atom on the phenyl ring; R¹ is hydrogen ormethyl; R² is hydrogen or methyl; R³ is hydrogen or methyl; R⁴ ishydrogen, methyl or ethyl; R⁵ is hydrogen or methyl; m is 1; and n is 1.

In another embodiment of compounds of formula (I), the As(OH)₂ group ispara- to the N-atom on the phenyl ring; R¹ is hydrogen; R² is hydrogenor methyl; R³ is hydrogen; R⁴ is hydrogen or methyl; R⁵ is hydrogen ormethyl; m is 1; and n is 1.

In a particular embodiment of the invention the compound of formula (I)is:

This compound is referred to herein as “Penicillamine-arsenoxide”.

In another embodiment of the invention the compound of formula (I) is:

This compound may be referred to herein as “cysteinyl-phenylarsenoxide”.

Synthesis of Compounds of Formula (I)

Compounds of formula (I) can be readily prepared by those skilled in theart using methods and materials known in the art and with reference tostandard text books, such as “Advanced Organic Chemistry” by Jerry March(third edition, 1985, John Wiley and Sons) or “Comprehensive OrganicTransformations” by Richard C. Larock (1989, VCH Publishers).

A representative scheme for the preparation of compounds of formula (I)is shown below:

-   -   where X is a leaving group and P¹ and P² are hydrogen or        protecting groups.

In Scheme 1 X is a leaving group capable of being displaced in anucleophilic reaction by a nucleophile. Suitable leaving groups includehalogens, such as iodo, bromo and chloro. Other suitable leaving groupswill be known to those skilled in the art. According to the presentinvention the nucleophilic group may be a thiol. The —SH may bedeprotonated by a base, such as sodium hydroxide, potassium hydroxide,sodium hydrogen carbonate, sodium carbonate, etc. The amino group and/orcarboxylic acid group may be protected. Suitable protecting groups areknown to those skilled in the art and reference may be had to“Protective Groups in Organic Synthesis” by Theodora Greene and PeterWuts (third edition, 1999, John Wiley and Sons).

In an alternative synthetic strategy, compounds of formula (I) accordingto the present invention may be prepared by enzymic cleavage of apeptidyl residue of an organo-arsenoxide compound. Suitable enzyme(s)can be selected depending on the composition of the peptidyl residue.Thus, for example, where an organo-arsenoxide starting compoundcomprises a tripeptide residue such as glutathione, compounds of formula(I) can be prepared by enzymic cleavage of the terminal γ-glutamylresidue with γ-glutamyl transpeptidase (e.g., ovine kidney γ-glutamyltranspeptidase type I), followed by cleavage of the glycinyl residuewith an aminopeptidase (e.g., porcine kidney aminopeptidase) to leave acysteinyl amino acid residue.

The stereochemistry at the chiral atom indicated by * in formula (I) maybe (R) or (S). The present invention includes enantiomerically pureforms of compounds of formula (I), mixtures of enantiomers in any ratio,as well as racemates. In one embodiment of the invention thestereochemistry at the chiral atom indicated by * in formula (I) is (R).In another embodiment the invention the stereochemistry at the chiralatom indicated by * in formula (I) is (S).

In another preferred embodiment of the invention the compound of formula(I) is (S)-Penicillamine-arsenoxide. In another preferred embodiment ofthe invention the compound of formula (I) is(R)-Penicillamine-arsenoxide. In another embodiment the compound offormula (I) comprises a mixture of (R) and (S) enantiomers ofPenicillamine-arsenoxide. In another embodiment, the mixture of (R) and(S) enantiomers of Penicillamine-arsenoxide is a racemic mixture.

In a preferred embodiment of the invention the compound of formula (I)is (S)-cysteinyl-phenylarsenoxide. In another preferred embodiment ofthe invention the compound of formula (I) is(R)-cysteinyl-phenylarsenoxide. In another embodiment the compound offormula (I) comprises a mixture of (R) and (S) enantiomers ofcysteinyl-phenylarsenoxide. In another embodiment, the mixture of (R)and (S) enantiomers of cysteinyl-phenylarsenoxide is a racemic mixture.

Also included within the scope of the present invention are allstereoisomers, geometric isomers and tautomeric forms of the compoundsof formula (I), including compounds exhibiting more than one type ofisomerism, and mixtures of one or more thereof. Also included are acidaddition or base salts wherein the counterion is optically active, forexample, d-lactate or l-lysine, or racemic, for example, dl-tartrate ordl-arginine.

Cis/trans (E/Z) isomers may be separated by conventional techniques wellknown to those skilled in the art, for example, chromatography andfractional crystallisation.

Conventional techniques for the preparation/isolation of individualenantiomers include chiral synthesis from a suitable optically pureprecursor or resolution of the racemate (or the racemate of a salt orderivative) using, for example, chiral high pressure liquidchromatography (HPLC).

Alternatively, the racemate (or a racemic precursor) may be reacted witha suitable optically active compound, for example, an alcohol, or, inthe case where the compound of formula I contains an acidic or basicmoiety, a base or acid such as 1-phenylethylamine or tartaric acid. Theresulting diastereomeric mixture may be separated by chromatographyand/or fractional crystallization and one or both of thediastereoisomers converted to the corresponding pure enantiomer(s) bymeans well known to a skilled person.

Chiral compounds of the invention (and chiral precursors thereof) may beobtained in enantiomerically-enriched form using chromatography,typically HPLC, on an asymmetric resin with a mobile phase consisting ofa hydrocarbon, typically heptane or hexane, containing from 0 to 50% byvolume of isopropanol, typically from 2% to 20%, and from 0 to 5% byvolume of an alkylamine, typically 0.1% diethylamine. Concentration ofthe eluate affords the enriched mixture.

Therapeutic Application(s)

Compounds of formula (I) in accordance with the present invention, andpharmaceutically acceptable salts and hydrates thereof, are capable ofbinding to cysteine residues of mitochondrial Adenine NucleotideTranslocator (ANT) in proliferating endothelial cells thereby inducingthe Mitochondrial Permeability Transition (MPT). Accordingly, compoundsof formula (I) according to the present invention may be used to induceproliferation arrest and cell death. Advantageously, compounds offormula (I) may selectively induce the MPT in proliferating endothelialcells, compared to other cells, such as tumor cells. Compounds offormula (I) may therefore be useful in the treatment of proliferativediseases.

Advantageously, compounds of formula (I), such asPenicillamine-arsenoxide and cysteinyl-phenylarsenoxide, may be moreeffective than known arsenoxide compounds, including organo-arsenoxidecompounds disclosed in WO 01/21628, such as the compound4-(N—(S-glutathionylacetyl)amino)phenylarsenoxide (“GSAO”), atinhibiting cellular proliferation (particularly proliferation ofendothelial cells) and/or reducing the viability of endothelial cells.In the context of this invention, “reducing the viability of endothelialcells” can include cell death, or progression towards cell death. Forexample, compounds of formula (I) may be about 5-times, about 10-times,about 15-times, about 20-times, about 25-times, about 30-times, about40-times, about 50-times, about 75-times, or about 100-times moreeffective than GSAO at inhibiting proliferation of endothelial cellsand/or reducing the viability of endothelial cells. In a particularembodiment, compounds of formula (I) are from about 5 to 50-times moreeffective than GSAO at inhibiting proliferation and/or reducing theviability of endothelial cells. In another embodiment, compounds offormula (I) are from about 10 to 30-times more effective than GSAO atinhibiting proliferation and/or reducing the viability of endothelialcells. In another embodiment, compounds of formula (I) are from about 20to 25-times more effective than GSAO at inhibiting proliferation and/orreducing the viability of endothelial cells.

Advantageously, compounds of formula (I), such asPenicillamine-arsenoxide and cysteinyl-phenylarsenoxide, may be moreefficient than known arsenoxide compounds, for example GSAO, at inducingthe Mitochondrial Permeability Transition (MPT). For example, the timefor half-maximal swelling of isolated mitochondria may be from about 2to about 20-times, about 2 to about 15-times, about 2 to about 10-times,about 2 to about 8-times, about 2 to about 6-times, or about 2 to about4-times faster for compounds of formula (I) compared to other arsenoxidecompounds, such as GSAO. In a particular embodiment of the inventioncompounds of formula (I) are from about 2 to about 10-times faster atinducing the MPT than other arsenoxide compounds, such as GSAO. Inanother embodiment, compounds of formula (I) are from about 2 to about6-times faster at inducing the MPT than other arsenoxide compounds, suchas GSAO. In another embodiment, compounds of formula (I) are from about4-times faster at inducing the MPT than other arsenoxide compounds, suchas GSAO.

The increased efficiency of inhibition of proliferating endothelialcells by compounds of formula (I) according to the present invention maybe due to increased accumulation in cells. For example, compounds offormula (I) may accumulate in endothelial cells at a faster rate incomparison to other arsenoxide compounds, such a GSAO. Accordingly,compounds of formula (I) may be more effective inhibitors of cellularproliferation than other organo-arsenoxide compounds, such as GSAO.

Thus, another embodiment of the invention relates to a method oftreating a cellular proliferative disease in a vertebrate, the methodcomprising administering to the vertebrate a therapeutically effectiveamount of at least one compound of formula (I) or a salt or hydratethereof, or a pharmaceutical composition thereof. The cells may beendothelial cells. Compounds of formula (I) may be selective forproliferating endothelial cells. Compounds of formula (I) may exhibitgreater selectivity for proliferating cells than the compound GSAO. Theproliferative disease may be cancer, such as solid tumors. Thus, aparticular embodiment of the invention relates to a method of treatingsolid tumours, the method comprising administering to the vertebrate atherapeutically effective amount of at least one compound of formula(I), or a salt or hydrate thereof, or a pharmaceutical compositionthereof. In preferred embodiments, the compound of formula (I) may bePenicillamine-arsenoxide or cysteinyl-phenylarsenoxide.

In another embodiment the present invention relates to a method ofinhibiting angiogenesis in a vertebrate, comprising administering to thevertebrate an effective amount of at least one compound of formula (I)or a salt or hydrate thereof, or a pharmaceutical composition thereof.

A further embodiment of the invention relates to a method of selectivelyinducing the MPT in proliferating cells in a vertebrate comprisingadministering to the vertebrate a therapeutically effective amount atleast one compound of formula (I) or a salt or hydrate thereof, or apharmaceutical composition thereof. Compounds of formula (I) accordingto the present invention may induce the MPT by binding to cysteineresidues on mitochondrial Adenine Nucleotide Translocator. The compoundof formula (I) may be from about 2 to about 20-times, about 2 to about10-times, about 2 to about 5-times, e.g., about 4-times, more efficientat inducing the MPT in proliferating cells than the compound GSAO.

Another embodiment of the invention relates to a method of inducingapoptosis in proliferating cells in a mammal, comprising administeringto the mammal an apoptosis-inducing amount of at least one compound offormula (I) or a salt or hydrate thereof, or a pharmaceuticalcomposition thereof. Compounds of formula (I) may selectively induceapoptosis in proliferating cells relative to normal cells. Compounds offormula (I) may be more effective at inducing apoptosis in proliferatingcells than the compound GSAO.

Compounds of formula (I) according to the present invention also havethe potential to be useful for treating acute promyelocytic leukaemia(APL). The current treatment of APL is all-trans retinoic acid (ATRA)therapy that targets the underlying molecular lesion and leads todifferentiation of leukaemic blasts into mature granulocytes (Reiter etal., 2004). However, treatment with ATRA is associated with the retinoicacid syndrome which can result in death. Relapse is also a problem. Inrelapsed patients, arsenic trioxide is considered the treatment ofchoice (Reiter et al., 2004). However, inorganic arsenicals, such asarsenic trioxide, have several disadvantages when used in therapy. Forexample, inorganic arsenicals, such as arsenic trioxide, have long beenrecognised as a poison and carcinogen when present in the body at levelsthat exceed its capacity to detoxify the metalloid. Arsenic trioxide isadministered by intravenous infusion over 2 h to minimize side effects,which include QTc prolongation, APL differentiation syndrome, peripheralneuropathies, hepatic dysfunction and gastrointestinal reactions (Evenset al., 2004). There is a need for safer arsenicals for the treatment ofAPL, including AML, and myelodysplastic syndrome.

Therefore, a further embodiment of the invention relates to a method oftreating leukaemia or myelodysplastic syndrome in a vertebrate,comprising administering to the vertebrate a therapeutically effectiveamount of at least one compound of formula (I) or a salt or hydratethereof, or a pharmaceutical composition thereof. In one embodiment theleukaemia is acute promyelocytic leukaemia (APL). In another embodimentthe leukaemia is acute myelocytic leukaemia (AML). In accordance withthe present invention, compounds of formula (I) may be at least aseffective as arsenic trioxide at inhibiting APL cells. In oneembodiment, compounds of formula (I) are more effective than arsenictrioxide in treating APL. Advantageously, compounds of formula (I) mayexhibit less side effects than arsenic trioxide. Compounds of formula(I) may be more effective than other organoarsenoxide compounds, such asGSAO, in treating APL, AML and/or myelodysplastic syndrome.

Another feature of compounds of formula (I) according to the presentinvention is that they may have reduced lipid solubility, for example,in comparison to arsenic trioxide. The water solubility of compounds offormula (I) is such that they may have reduced penetration into tissuesand be mostly restricted to the intravascular compartment. Therefore,compounds of formula (I) may advantageously may result in less sideeffects than other arsenicals, such as arsenic trioxide.

Therapeutic advantages may be realised through combination regimens. Incombination therapy the respective agents may be administeredsimultaneously, or sequentially in any order. Accordingly, methods oftreatment according to the present invention may involve administrationof one or more compounds of formula (I). Compound(s) of formula (I) maybe administered in conjunction with conventional therapy, such asradiotherapy, chemotherapy, surgery, or other forms of medicalintervention. Examples of chemotherapeutic agents include adriamycin,taxol, fluorouricil, melphalan, cisplatin, oxaliplatin, alphainterferon, vincristine, vinblastine, angioinhibins, TNP-470, pentosanpolysulfate, platelet factor 4, angiostatin, LM-609, SU-101, CM-101,Techgalan, thalidomide, SP-PG and the like. Other chemotherapeuticagents include alkylating agents such as nitrogen mustards includingmechloethamine, melphan, chlorambucil, cyclophosphamide and ifosfamide,nitrosoureas including carmustine, lomustine, semustine andstreptozocin; alkyl sulfonates including busulfan; triazines includingdicarbazine; ethyenimines including thiotepa and hexamethylmelamine;folic acid analogues including methotrexate; pyrimidine analoguesincluding 5-fluorouracil, cytosine arabinoside; purine analoguesincluding 6-mercaptopurine and 6-thioguanine; antitumour antibioticsincluding actinomycin D; the anthracyclines including doxorubicin,bleomycin, mitomycin C and methramycin; hormones and hormone antagonistsincluding tamoxifen and cortiosteroids and miscellaneous agentsincluding cisplatin and brequinar, and regimens such as COMP(cyclophosphamide, vincristine, methotrexate and prednisone), etoposide,mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide,vincristine and dexamethasone), and PROMACE/MOPP (prednisone,methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol,etoposide/mechlorethamine, vincristine, prednisone and procarbazine).

Pharmaceutical and/or Therapeutic Formulations

Typically, for medical use, salts of the compounds of the presentinvention will be pharmaceutically acceptable salts; although othersalts may be used in the preparation of the inventive compounds or ofthe pharmaceutically acceptable salt thereof. By pharmaceuticallyacceptable salt it is meant those salts which, within the scope of soundmedical judgement, are suitable for use in contact with the tissues ofhumans and lower animals without undue toxicity, irritation, allergicresponse and the like, and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are well known inthe art.

Pharmaceutically acceptable salts of compounds of formula I may beprepared by methods known to those skilled in the art, including forexample, (i) by reacting a compound of formula I with the desired acidor base; (ii) by removing an acid- or base-labile protecting group froma suitable precursor of the compound of formula I or by ring-opening asuitable cyclic precursor, for example, a lactone or lactam, using thedesired acid or base; or (iii) by converting one salt of the compound offormula I to another by reaction with an appropriate acid or base or bymeans of a suitable ion exchange column.

All three reactions are typically carried out in solution. The resultingsalt may precipitate out and be collected by filtration or may berecovered by evaporation of the solvent. The degree of ionisation in theresulting salt may vary from completely ionised to almost non-ionised.

Thus, for instance, suitable pharmaceutically acceptable salts ofcompounds according to the present invention may be prepared by mixing apharmaceutically acceptable acid such as hydrochloric acid, sulfuricacid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid,benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid,tartaric acid, or citric acid with the compounds of the invention.Suitable pharmaceutically acceptable salts of the compounds of thepresent invention therefore include acid addition salts.

S. M. Berge et al. describe pharmaceutically acceptable salts in detailin J. Pharmaceutical Sciences, 1977, 66:1-19. The salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention, or separately by reacting the free base function with asuitable organic acid. Representative acid addition salts includeacetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, digluconate, cyclopentanepropionate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium potassium, calcium, magnesium, and the like, as well asnon-toxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,triethanolamine and the like.

Convenient modes of administration include injection (subcutaneous,intravenous, etc.), oral administration, inhalation, transdermalapplication, topical creams or gels or powders, or rectaladministration. In one embodiment, the mode of administration isparenteral. In another embodiment, the mode of administration is oral.Depending on the route of administration, the formulation and/orcompound may be coated with a material to protect the compound from theaction of enzymes, acids and other natural conditions which mayinactivate the therapeutic activity of the compound. The compound alsomay be administered parenterally or intraperitoneally.

Dispersions of compounds according to the invention may also be preparedin glycerol, liquid polyethylene glycols, and mixtures thereof and inoils. Under ordinary conditions of storage and use, pharmaceuticalpreparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. Ideally, the composition is stable under theconditions of manufacture and storage and may include a preservative tostabilise the composition against the contaminating action ofmicroorganisms such as bacteria and fungi.

The compound(s) of the invention may be administered orally, forexample, with an inert diluent or an assimilable edible carrier. Thecompound(s) and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto an individual's diet. For oral therapeutic administration, thecompound(s) may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Suitably, such compositionsand preparations may contain at least 1% by weight of active compound.The percentage of the compound(s) of formula (I) in pharmaceuticalcompositions and preparations may, of course, be varied and, forexample, may conveniently range from about 2% to about 90%, about 5% toabout 80%, about 10% to about 75%, about 15% to about 65%; about 20% toabout 60%, about 25% to about 50%, about 30% to about 45%, or about 35%to about 45%, of the weight of the dosage unit. The amount of compoundin therapeutically useful compositions is such that a suitable dosagewill be obtained.

The language “pharmaceutically acceptable carrier” is intended toinclude solvents, dispersion media, coatings, anti-bacterial andanti-fungal agents, isotonic and absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the compound, use thereof in thetherapeutic compositions and methods of treatment and prophylaxis iscontemplated. Supplementary active compounds may also be incorporatedinto the compositions according to the present invention. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. “Dosageunit form” as used herein refers to physically discrete units suited asunitary dosages for the individual to be treated; each unit containing apredetermined quantity of compound(s) is calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The compound(s) may be formulated for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in an acceptable dosage unit. In thecase of compositions containing supplementary active ingredients, thedosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

In one embodiment, the carrier is an orally administrable carrier.

Another form of a pharmaceutical composition is a dosage form formulatedas enterically coated granules, tablets or capsules suitable for oraladministration.

Also included in the scope of this invention are delayed releaseformulations.

Compounds of formula (I) according to the invention also may beadministered in the form of a “prodrug”. A prodrug is an inactive formof a compound which is transformed in vivo to the active form. Suitableprodrugs include esters, phosphonate esters etc, of the active form ofthe compound.

In one embodiment, the compound of formula (I) may be administered byinjection. In the case of injectable solutions, the carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby including various anti-bacterial and/or anti-fungal agents. Suitableagents are well known to those skilled in the art and include, forexample, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid,thimerosal, and the like. In many cases, it may be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theanalogue in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the analogue into a sterile vehicle which contains a basicdispersion medium and the required other ingredients from thoseenumerated above.

Tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum gragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin or a flavouring agent such as peppermint,oil of wintergreen, or cherry flavouring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the analogue, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavouring such ascherry or orange flavour. Of course, any material used in preparing anydosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the analogue can beincorporated into sustained-release preparations and formulations.

Preferably, the pharmaceutical composition may further include asuitable buffer to minimise acid hydrolysis. Suitable buffer agentagents are well known to those skilled in the art and include, but arenot limited to, phosphates, citrates, carbonates and mixtures thereof.

Single or multiple administrations of the compounds and/orpharmaceutical compositions according to the invention may be carriedout. One skilled in the art would be able, by routine experimentation,to determine effective, non-toxic dosage levels of the compound and/orcomposition of the invention and an administration pattern which wouldbe suitable for treating the diseases and/or infections to which thecompounds and compositions are applicable.

Further, it will be apparent to one of ordinary skill in the art thatthe optimal course of treatment, such as the number of doses of thecompound or composition of the invention given per day for a definednumber of days, can be ascertained using convention course of treatmentdetermination tests.

Generally, an effective dosage per 24 hours may be in the range of about0.0001 mg to about 1000 mg per kg body weight; for example, about 0.001mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg perkg body weight; about 0.1 mg to about 500 mg per kg body weight; about0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250mg per kg body weight. More suitably, an effective dosage per 24 hoursmay be in the range of about 1.0 mg to about 200 mg per kg body weight;about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about50 mg per kg body weight; about 1.0 mg to about 25 mg per kg bodyweight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg toabout 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kgbody weight.

Alternatively, an effective dosage may be up to about 500 mg/m². Forexample, generally, an effective dosage is expected to be in the rangeof about 25 to about 500 mg/m², about 25 to about 350 mg/m², about 25 toabout 300 mg/m², about 25 to about 250 mg/m², about 50 to about 250mg/m², and about 75 to about 150 mg/m².

In another embodiment, a compound of Formula (I) may be administered inan amount in the range from about 100 to about 1000 mg per day, forexample, about 200 mg to about 750 mg per day, about 250 to about 500 mgper day, about 250 to about 300 mg per day, or about 270 mg to about 280mg per day.

Compounds in accordance with the present invention may be administeredas part of a therapeutic regimen with other drugs. It may desirable toadminister a combination of active compounds, for example, for thepurpose of treating a particular disease or condition. Accordingly, itis within the scope of the present invention that two or morepharmaceutical compositions, at least one of which contains a compoundof formula (I) according to the present invention, may be combined inthe form of a kit suitable for co-administration of the compositions.

The invention will now be described in more detail, by way ofillustration only, with respect to the following examples. The examplesare intended to serve to illustrate this invention and should not beconstrued as limiting the generality of the disclosure of thedescription throughout this specification.

EXAMPLES Example 1 Preparation and Efficacy of(S)-Penicillamine-Arsenoxide (“PENAO”)

Materials and Methods

Synthesis and Purification of (S)-Penicillamine-Arsenoxide:

p-Arsanilic acid (10 g, 46.07 mmol) was dissolved in a 1.18 M Na₂CO₃solution, made from Na₂CO₃ (15 g, 141.5 mmol) dissolved in H₂O (120 mL)in a 500 mL round-bottom flask. The solution was cooled in a 4° C.fridge for 2 hours and then placed in an ice bath upon a magneticstirrer. A solution of bromoacetyl bromide (9 mL, 101.4 mmol) in CH₂Cl₂(14 mL) was added in 4 aliquots to the flask while the mixture wasvigorously stirred. Addition took about 1 min with CO₂ evolution. Themixture was allowed to stir in the ice-bath for 5 min, and then at roomtemperature for 30 min till CO₂ evolution ceased. The mixture wasdecanted into a 250 mL separatory funnel. Additional CH₂Cl₂ (10 mL) wasadded and the layers were allowed to separate for about 10 min. TheCH₂Cl₂ layer was separated and the aqueous layer was placed in a 400 mLbeaker. The solution was stirred and acidified with 98% H₂SO₄ (2.8 mL)to pH 4. A white precipitate resulted which was collected by filtration(14.83 g, 95% yield). The resulting 4-(2-bromoacetylamino)benzenearsonicacid (“BRAO”) (14.83 g, 43.88 mmol) was dissolved in 1:1 HBr/MeOH (210mL) in a 500 mL 3-neck round bottom flask. NaI (5 mg) was added and themixture was stirred. SO₂ was bubbled through at ca. 2 bubbles/second andafter 10 min a white precipitate started to form. SO₂ was bubbledthrough for a further 20 h and the mixture was stirred at a mediumspeed. The solid was collected by filtration, washed with the filtratethen water (30 mL×3), and placed on the rotary evaporator at 50° C. for5 h to give 4-(2-bromoacetylamino)benzenearsonous acid (6.04 g, 38.8%yield). A portion of the 4-(2-bromoacetylamino)benzenearsonous acid (500mg, 1.553 mmol) was dissolved in nitrogen-flushed DMSO (10 mL) and addeddrop-wise over about 1 min to an solution of S-penicillamine (265 mg,1.77 mmol) in an aqueous NaHCO₃ solution (840 mg, 10 mmol) which usednitrogen-saturated H₂O (20 mL). The addition took place in a 100 mLround-bottom flask and the clear solution was stirred on a low speedunder argon for 4 h. The solution was acidified with 98% H₂SO₄ (about0.2 mL) to pH 5. Acetone (500 mL) was stirred vigorously, and theacidified solution was added drop-wise over about 5 min to yield a whiteprecipitate. The supernatant was centrifuged, decanted, and theresulting white solid was further washed and re-centrifuged with acetone(20 mL×2), transferred with acetone (40 mL) into a 100 mL pear-shapedflask and dried on the rotary evaporator at 25° C. for 2 h. CrudePenicillamine-arsenoxide was found to be about 30% pure by internalstandard ¹H-NMR.

Crude (S)-Penicillamine-arsenoxide (100.2 mg, 0.077 mmol as 30% pure)was dissolved in nitrogen-saturated H₂O (2.5 mL) and purified on a LowPressure Liquid Chromatography system. The conditions used were a 30 cmcolumn with a 1.25 cm internal radius, nitrogen-saturated H₂O as therunning buffer, Biogel P-2 resin and a rate of 0.25 mL/min. The secondpeak was collected in a 50 mL Falcon tube, frozen in liquid N₂,freeze-dried for 3 days, and placed in a desiccator for 1 day to yielddried pure (S)-Penicillamine-arsenoxide (20.3 mg, 0.052 mmol). Theprocess was repeated with more portions of crudePenicillamine-arsenoxide (696 mg in total) and this yielded purified(S)-Penicillamine-arsenoxide (114 mg, 26.5% yield). The structure of(S)-Penicillamine-arsenoxide (FIG. 1) was confirmed by MS, ¹H-NMR and 2DNMR. The purity obtained was 90% by an arsenical activity assay. Themain impurity was water as the final product is extremely hygroscopic.The molecular weight of (S)-Penicillamine-arsenxoide is 390.28 g/mole.

¹H-NMR (300 MHz, D₂O): δ 1.32 (s, 3H), 1.53 (s, 3H), 3.55 (d, J=3.4 Hz,2 H), 3.63 (s, 1H), 7.52 (d, J=8.3 Hz, 2H), 7.68 (d, J=8.3 Hz, 2H). Theproton NMR spectrum (FIG. 2) was recorded on a Bruker, dual channelprobe NMR spectrometer. Rapid keto-enol tautomerism and subsequentdeuterium replacement results in the loss of the doublet peak at δ3.5518 which occurs over 1 h. This can be monitored using time-dependantNMR.

¹³C-NMR (D₂O): δ 23.15, 26.83, 33.12, 46.75, 61.36, 121.62, 130.04, 139,144, 170.

The structure of (S)-Penicillamine-arsenoxide was also confirmed by anHMBC experiment (2D ¹H-¹³C multiple bond coupling, see FIG. 3).

MS: m/z 413.011678 (M+Na)⁺ (C₁₃H₁₉SO₅N₂AsNa requires 413.012285). (FIG.4)

Synthesis of Penicillamine-Arsonic Acid

p-Arsanilic acid (10 g, 46.07 mmol) was dissolved in a 1.18 M Na₂CO₃solution, made from Na₂CO₃ (15 g, 141.5 mmol) dissolved in H₂O (120 mL)in a 500 mL round-bottom flask. The solution was cooled in a 4° C.fridge for 2 hours and then placed in an ice bath upon a magneticstirrer. A solution of bromoacetyl bromide (9 mL, 101.4 mmol) in CH₂Cl₂(14 mL) was added in 4 aliquots to the flask while the mixture wasvigorously stirred. Addition took about 1 min with CO₂ evolution. Themixture was allowed to stir in the ice-bath for 5 min, and then at roomtemperature for 30 min till CO₂ evolution ceased. The mixture wasdecanted into a 250 mL separatory funnel. Additional CH₂Cl₂ (10 mL) wasadded and the layers were allowed to separate for about 10 min. TheCH₂Cl₂ layer was separated and the aqueous layer was placed in a 400 mLbeaker. The solution was stirred and acidified with 98% H₂SO₄ (2.8 mL)to pH 4. The resulting white precipitate4-(2-bromoacetylamino)benzenearsonic acid (“BRAO”) was collected byfiltration (14.83 g, 95% yield).

A portion of 4-(2-bromoacetylamino)benzenearsonic acid (500 mg, 1.479mmol) was dissolved in aqueous NaHCO₃ solution (420 mg, 4.999 mmol) inH₂O (10 mL) and added drop-wise over about 1 min to a solution of(S)-penicillamine (265 mg, 1.77 mmol) in an aqueous NaHCO₃ solution (640mg, 7.618 mmol) in H₂O (15 mL). The addition took place in a 100 mLround-bottom flask and the clear solution was stirred on a low speed for4 h. The solution was acidified with 98% H₂SO₄ (about 0.25 mL) to pH 5.A 1:1 acetone:ethanol (500 mL) solution was stirred vigorously, and theacidified solution was added drop-wise over about 5 min to yield a whiteprecipitate. The supernatant was centrifuged, which was decanted, andthe resulting white solid was further washed and re-centrifuged with 1:1acetone:ethanol (25 mL×2), transferred with 1:1 acetone:ethanol (50 mL)into a 100 mL pear-shaped flask and dried on the rotary evaporator at25° C. for 2 h. The resulting (S)-Penicillamine-arsonic acid was foundto be about 44% pure by internal standard ¹H-NMR spectroscopy and wasused without further purification (1.022 g, 75% yield). The structure of(S)-Penicillamine-arsonic acid was confirmed by MS, ¹H-NMR and 2D NMR.The main impurity was water as the final product is extremelyhygroscopic. The molecular weight is 406.28 g/mole.

GSAO was prepared as previously described (Don et al., 2003).

A 1 M solution of arsenic trioxide was prepared by dissolving the solid(Sigma, St. Louis, Mo.) in 3 M NaOH prepared in deoxygenated water. Thesolution was diluted 10-fold in deoxygenated water, the pH adjusted to7.0 using HCl and stored at 4° C. in an airtight container until use.

Arsenical Assay

(S)-Penicillamine-arsenoxide was dissolved in the titration buffer,sterile filtered, and the concentration determined by titrating withdimercaptopropanol and calculating the remaining free thiols with5,5′-dithiobis(2-nitrobenzoic acid). The solution was stored at 4° C. inthe dark until use. There was no significant loss in the activeconcentration of stock solutions of the arsenicals for at least a monthwhen stored under these conditions.

Mitochondrial Swelling Assay

Mitochondria were isolated from the livers of ˜250 g male Wistar ratsusing differential centrifugation as previously described (Dilda et al.,2005a; Don et al., 2003). The final mitochondrial pellet was resuspendedin 3 mM HEPES-KOH, pH 7.0 buffer containing 213 mM mannitol, 71 mMsucrose and 10 mM sodium succinate at a concentration of 30 mg ofprotein per mL. Mitochondrial permeability transition induction wasassessed spectrophotometrically by suspending the liver mitochondria at0.5 mg of protein per mL at 25° C. in 3 mM HEPES-KOH, pH 7.0 buffercontaining 75 mM mannitol, 250 mM sucrose, 10 mM sodium succinate, and 2μM rotenone. Swelling was measured by monitoring the associated decreasein light scattering at 520 nm using a SpectraMax Plus microplate reader(Molecular Devices, Palo Alto, Calif.).

Cell Culture

BAE cells were from Cell Applications, San Diego, Calif. and BxPC-3,HT1080, LLC, PANC-1, MCF-7, HCT116 and K562 cells were from ATCC,Bethesda, Md. NB4 and MDCK2 cells were from Shane Supple (KanematsuLaboratories, Royal Prince Alfred Hospital, Sydney, Australia) and P.Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands).BAE, HT1080, Panc-1, MCF-7, HCT116, MDCK2 and LLC cells were cultured inDMEM. NB4, K562 and BxPC-3 cells were cultured in RPMI medium. The cellswere supplemented with 10% foetal calf serum (FBS), 2 mM L-glutamine,and 1 U.mL-1 penicillin/streptomycin. Cell culture plasticware was fromTechno Plastic Products (Trasadingen, Switzerland). All other cellculture reagents were from Gibco (Gaithersburg, Md.).

Cell Proliferation and Viability Assays.

BAE, NB4, K562, MDCK2, HT1080, LLC, HCT116, Panc-1, MCF-7 and BxPC-3cells were seeded at a density of 1.5×10³, 3×10³, 4×10³, 5×10², 2×10³,5×10², 5×10², 6×10³, 6×10³ and 1×10⁴ cells per well, respectively, into96-well plates. Adherent cells were allowed to adhere overnight. Theywere then cultured for 72 h in medium containing 10% fetal calf serumand (S)-Penicillamine-arsenoxide. Viable cells were determined byincubating cells with the tetrazolium salt MTT (Sigma, St. Louis, Mo.),which is metabolized by viable cells to form insoluble purple formazancrystals. DMSO was added to lyse cells, the contents of the wells werehomogenized and the absorbance measured at 550 nm. Cell number in theuntreated control was normalized as 100%, and viable cell number for alltreatments was expressed as percentage of control. The cytotoxic effectsof (S)-Penicillamine-arsenoxide were assayed by flow cytometry withpropidium iodide. BAE cells were seeded at a density of 5×10⁴ cells perwell into 12-well plates, allowed to adhere overnight, then treated for48 h with GSAO. Adherent cells were detached with trypsin/EDTA andcombined with the growth medium containing the cells that had detachedduring treatment. The combined cells were pelleted, resuspended in 0.5mL serum-free medium containing 1 μg.mL⁻¹ propidium iodide (MolecularProbes, Eugene, Oreg.) and analysed by flow cytometry.

Flux of (S)-Penicillamine-arsenoxide.

BAE cells were seeded at a density of 1.5×10⁶ cells in Petri dishes andallowed to adhere overnight. Cells were incubated with 50 μM(S)-Penicillamine-arsenoxide at discrete times for up to 2 h at 37° C.and then washed three times with ice-cold PBS. The washed cells werelysed in 1 mL of 70% w/w nitric acid. Petri dishes were then washedtwice with 1 mL of PBS and kept at 4° C. until use. Samples were diluted10-fold and analysed for arsenic atoms using an Elan 6100 InductivelyCoupled Plasma Spectrometer (Perkin Elmer Sciex Instruments, Shelton,Conn.).

Organic Anion Transporting Polypeptide (OATP) studies. 750,000 BAE cellswere seeded in 6 well plates containing DMEM with 10% fetal calf serumand allowed to adhere overnight. Cells were pretreated or not with 500μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) for 30 minand then incubated with 20 μM (S)-Penicillamine arsenoxide for 2 h at37° C. and 5% CO₂. Cells were then washed twice with ice-cold PBS andlysed with 70% nitric acid. Cellular arsenic levels were determined byICPMS.

5000 BAE cells were seeded in 96 well plates containing DMEM with 10%fetal calf serum and allowed to adhere overnight. Cells were pretreatedor not with 300 μM DIDS for 30 min and then incubated with 1.5 μM(S)-Penicillamine arsenoxide for 24 h at 37° C. and 5% CO₂. Cellviability was determined using MTT.

Drug transporter transfectants. Transfectants of the Madin-Darby caninekidney II (MDCKII) polarised epithelial cell line, over-expressingmultidrug resistance-associated proteins (MRP) 1, 2 or 3, have beendescribed (Evers et al., 2000; Kool et al., 1999), as have the MEF/MDR1clone H4 over-expressing human MDR1 or BCRP (Dilda et al., 2005b). Cellswere grown and maintained as adherent monolayers in DMEM containing 10%calf serum (Cosmic™, Hyclone, Tauranga, New Zealand), 100 μg.mL⁻¹penicillin and 60 μg.mL⁻¹ streptomycin. Cytotoxicity assays wereperformed as described previously (Allen et al., 1999).

Primary tumor growth assays. Female 7 to 9 week old Balb C nude micewere used (UNSW Biological Resource Centre). Mice were held in groups of3 to 5 at a 12 h day and night cycle and were given animal chow andwater ad libidum. A suspension of 2×10⁶ BxPC-3 cells in 0.2 mL of PBSwas injected subcutaneously in the proximal midline. Tumors were allowedto establish and grow to a size of ˜50 mm³ after which they wererandomized into four groups. Tumor volume was calculated using therelationship length×height×width×0.523. Tumor doubling time (T_(D)) wascalculated from the tumor growth rate curve during exponential growthusing the formula T_(D)=0.693/ln(V_(F)/V_(I)), where V_(F) is finaltumor volume and V_(I) is initial tumor volume (Wolff et al., 2004).Animals were implanted with 28 day alzet model 1004 micro-osmotic pumps(ALZA Corporation, Palo Alto, Calif.) subcutaneously in the flank. Thepumps delivery 0.25, 0.5 or 1 mg/kg/day (S)-Penicillamine arsenoxide in100 mM glycine. Tumor volume and animal weight was measured every 2 or 3days.

Statistical Analyses.

Results are presented as means±SD. All tests of statistical significancewere two-sided, and P values<0.05 were considered statisticallysignificant.

Results and Discussion

(S)-Penicillamine-Arsenoxide Inhibits Proliferation of Mammalian Cells

Reported IC₅₀ values for proliferation arrest and loss of viability ofbovine aortic endothelial cells (BAE) cells induced by GSAO are 10 μMand 75 μM, respectively (Dilda et al., 2005a; Don et al., 2003). TheIC₅₀ for proliferation arrest of BAE cells is 0.4 μM for(S)-Penicillamine-arsenoxide (FIG. 5) compared to 10 μM for GSAO, whilethe IC₅₀ value for loss of viability is 3.5 μM (FIG. 6).(S)-Penicillamine-arsenoxide, therefore, is ˜25-times more effectivethan GSAO at blocking proliferation and reducing the viability ofendothelial cells.

(S)-Penicillamine-arsenoxide is a selective inhibitor of endothelialcells compared to tumour cells. Comparison of the IC₅₀ for proliferationarrest of endothelial and epithelial cells compared to eight differenttumour cell lines is shown in Table 1. All tumour cells tested were 1.6to 30-fold more resistant to (S)-Penicillamine-arsenoxide thanendothelial cells. BAE cells were also 5.6-fold more sensitive to(S)-Penicillamine-arsenoxide than kidney epithelial cells.(S)-Penicillamine-arsenoxide is equivalent to arsenic trioxide in itseffects on APL cells, while GSAO is ˜10-fold less active (FIG. 7).

TABLE 1 (S)-Penicillamine-arsenoxide IC₅₀ values for proliferationarrest for various cell lines. Cell Type Cell Line IC₅₀, μM bovineaortic endothelial BAEC 0.43 human acute promyelocytic leukaemia NB40.70 human chronic myelogenous leukaemia K562 1.4 dog kidney epithelialMDCK2 2.4 human fibrosarcoma HT1080 4.0 human lung carcinoma LLC 5.0human colorectal carcinoma HCT1116 6.0 human pancreatic carcinoma PANC-16.5 human mammary carcinoma MCF-7 9.0 human pancreatic carcinoma BxPC-313

(S)-Penicillamine-arsenoxide is also more efficient than GSAO atinducing the mitochondrial permeability transition.(S)-Penicillamine-arsenoxide, like GSAO, triggered swelling of isolatedrat liver mitochondria in a time- and concentration-dependent manner(FIG. 8). The time for half-maximal swelling of isolated mitochondriawas approximately 4 times faster for (S)-Penicillamine-arsenoxidecompared to GSAO.

Without intending to be bound by any particular theory, a possiblemechanism for the increased anti-proliferative activity of(S)-Penicillamine-arsenoxide compared to GSAO was increased accumulationin cells. This theory was tested by comparing the uptake of the twocompounds in endothelial cells by measuring cellular accumulation ofarsenic. (S)-Penicillamine-arsenoxide accumulated in BAE cells at anapproximately 70-fold faster rate than GSAO (FIG. 9). The initial ratesof accumulation of GSAO and (S)-Penicillamine-arsenoxide were 1 and 69μmol per 10⁶ cells per min, respectively.

OATP is Involved in (S)-Penicillamine Transport Across the PlasmaMembrane

DIDS is an inhibitor of the plasma membrane organic anion transportingpolypeptide (OATP) (Kobayashi et al., 2003). The finding that thiscompound inhibits (S)-Penicillamine arsenoxide uptake (FIG. 10A) andreduces its anti-proliferative activity (FIG. 10B) in BAE cells impliesthat this transporter is involved in (S)-Penicillamine arsenoxide uptakeinto these cells.

(S)-Penicillamine Arsenoxide is Exported from the Cell by MRP1 and 2

MRP1/2 mediates export of GSAO from BAE cells (Dilda et al., 2005b).Penicillamine-arsenoxide is also a substrate for MRP1/2. More(S)-Penicillamine-arsenoxide accumulated in BAE cells in the presence ofthe MRP1/2 inhibitors 4H10 and MK-571 (FIG. 11A), which correlated withmore potent anti-proliferative effect (FIG. 11B). The inhibitors alonehad no effect on BAE cell proliferation (data not shown).

Mammalian cells over-expressing MRP1, 2, 3 or 6, or MDR1 or BCRP weretested for resistance to (S)-Penicillamine-arsenoxide. MRP1, MRP2 orMRP3 was over-expressed in the canine kidney epithelial MDCKII cellline, while MRP6, MDR1 or BCRP was over-expressed in the murine embryofibroblast MEF3.8 line. Cells were exposed to the indicatedconcentrations of (S)-Penicillamine-arsenoxide for 96 h and the numberof viable cells measured and expressed relative to the number ofuntreated cells. Resistance factor is calculated relative to the(S)-Penicillamine arsenoxide IC₅₀ for proliferation arrest ofnon-transfected parental cells.

TABLE 2 Resistance of mammalian cells over-expressing different drugtransporters to (S)-Penicillamine-arsenoxide. Transporter ResistanceFactor MD/MRP1 3.7 MD/MRP2 4.6 MD/MRP3 0.8 MEF/MRP6 1.3 MEF/MDR1 1.2MEF/BCRP 1.1

These results indicate that both GSAO and (S)-Penicillamine-arsenoxideare exported from BAE cells by MRP1/2.

Treatment of BAE cells with glutathione reduced GSAO inhibition of BAEcell proliferation, while blocking de novo synthesis of glutathione withbuthionine sulfoximine (BSO), an inhibitor of the γ-glutamyl cysteinesynthase, enhanced the proliferation arrest by almost 100-fold (Dilda etal., 2005b). These results indicated that MRP1/2 requires cellularglutathione for efficient transport of GSAO from the cell. Similar tothe findings with GSAO, treating BAE cells with BSO enhanced the(S)-Penicillamine-arsenoxide IC₅₀ for proliferation arrest byapproximately 25-fold (FIG. 12).

These results indicate that (S)-Penicillamine-arsenoxide is a moreeffective inhibitor of endothelial cells because it accumulates in thecells at a much faster rate than GSAO.

Anti-Tumour Activity of (S)-Penicillamine Arsenoxide

BalbC nude mice bearing subcutaneous human BxPC-3 pancreatic carcinomatumours in the proximal midline were implanted with 28 day micro-osmoticalzet pumps subcutaneously in the flank. The pumps delivered 0.25, 0.5or 1 mg per kg per day (S)-Penicillamine arsenoxide. The growth of theBxPC-3 tumours was significantly inhibited in the mice receiving(S)-Penicillamine arsenoxide (FIG. 13). The tumour doubling times are9.2, 8.3, 13.9 and 16.2 days for groups treated with vehicle (100 mMglycine) or 0.25, 0.5 and 1 mg/kg/day (S)-Penicillamine arsenoxide,respectively.

There was no change in the weight of the vehicle-versus(S)-Penicillamine arsenoxide-treated animals (not shown). There was someskin toxicity at the pump delivery site in the highest dose animals.There was skin necrosis at the delivery site in 3 of the 10 mice and in1 mouse there was an accumulation of connective tissue. There was someevidence of accumulation of connective tissue at the delivery site inthe occasional mouse at the lower doses of (S)-Penicillamine arsenoxide.

Example 2 Preparation and Efficacy of4-(N—(S-Cysteinylacetyl)amino)-phenylarsinous acid (“CAO”)

Materials and Methods

Cell Proliferation Assay

Bovine aortic endothelial (BAE) cells were from Cell Application (SanDiego, Calif.). BAE cells were cultured in DMEM supplemented with 10%fetal calf serum, 2 mM L-glutamine, and 5 units per mL penicillin andstreptomycin (Gibco, Gaithersburg, Md.). Cells were cultured at 37° C.in a 5% CO₂, 95% air atmosphere. BAE cells were seeded in 96-well plates(5,000 cells per well) in 0.2 ml of culture medium. After 24 h ofgrowth, the medium was replaced with fresh culture medium supplementedwith GSAO, CAO or 4H10 and cells were cultured for an additional 24, 48or 72 h. Viable attached cells were determined using the tetrazoliumsalt MTT (Sigma, St. Louis, Mo.) according to the manufacturer'sprotocol. Results were expressed as percentage of untreated controls.

Preparation of CAO

GSAO was produced as described previously (WO 01/21628) to a purity >94%by HPLC. A 50 mM solution of GSAO was made by dissolving solid in 20 mMHepes, pH 7.0 buffer containing 0.14 M NaCl, 20 mM glycine and 1 mMEDTA. 4-(N—(S-cysteinylglycylacetyl)amino)phenylarsinous acid wasproduced by cleaving the γ-glutamyl group from GSAO with ovine kidneyγ-glutamyl transpeptidase type I (Sigma, product number G8040) (FIG.14). A 10 mM solution of GSAO was incubated with 0.55 units per ml γGTin 15 mM Tris, pH 7.4 buffer containing 40 mM glycyl-glycine for 1 h at30° C. The γGT was removed from the reaction by filtration using a YM3Microcon membrane (Millipore, Billerica, Mass.).

4-(N—(S-cysteinylacetyl)amino)phenylarsinous acid (CAO) was produced bycleaving the glycine amino acid from4-(N—(S-cysteinylglycylacetyl)amino)phenylarsinous acid with porcinekidney aminopeptidase N (Type IV-S, Sigma, product number L5006) (FIG.14). The filtrate was incubated with 2 units per ml aminopeptidase N for1 h at 37° C. The aminopeptidase N was removed from the reaction byfiltration using a YM3 Microcon membrane (Millipore). The concentrationof CAO was measured by titrating with dimercaptopropanol and calculatingthe remaining free thiols with 5,5′-dithiobis(2-nitrobenzoic acid) (Donet al., 2003). The titrated solutions were sterile filtered and storedat 4° C. in the dark until use.

HPLC Analysis

GSAO and CAO were characterized by HPLC (1200 Series; AgilentTechnologies, Santa Clara, Calif.). Samples were resolved on a ZorbaxEclipse XDB-C18 column (4.6×150 mm, 5 μm; Agilent Technologies) using amobile phase of acetonitrile-water (25:75 vol/vol), flow rate of 0.5ml.min⁻¹ and detection by absorbance at 256 nm (FIG. 15).

Accumulation of GSAO and CAO in BAE Cells

Depending on the type of experiments, 1.6×10⁶ or 7.5×10⁵ BAE cells wereseeded in petri dishes or 6-well-plates, respectively, and allowed toattach overnight. The medium was replaced and the cells were incubatedfor 30 min in the absence or presence of acivicin or 4H10. The cellswere then incubated with 50 or 100 μM GSAO or CAO for 30 min for 4 h.Cells were then washed twice with ice-cold PBS and lysed with 1 ml of70% w/w nitric acid. Lysates were diluted 30-fold and analyzed forarsenic atoms using an Elan 6100 Inductively Coupled Plasma Spectrometer(Perkin Elmer Sciex Instruments, Shelton, Conn.).

Mitochondrial swelling assay. Mitochondria were isolated from the liversof ˜20 g female BalbC nude mice using differential centrifugation asdescribed previously (Dilda et al., 2005a). The final mitochondrialpellet was resuspended in 3 mM Hepes-KOH, pH 7.0 buffer containing 213mM mannitol, 71 mM sucrose and 10 mM sodium succinate at a concentrationof 30 mg of protein per mL. Mitochondrial permeability transitioninduction was assessed spectrophotometrically by suspending the livermitochondria at 1 mg of protein per ml at 37° C. in 3 mM Hepes-KOH, pH7.0 buffer containing 75 mM mannitol, 250 mM sucrose, 10 mM sodiumsuccinate, and 2 mM rotenone (Dilda et al., 2005a). Swelling wasmeasured by monitoring the associated decrease in light scattering at520 nm using a Molecular Devices M2 Microplate Reader (Palo Alto,Calif.).

Results and Discussion

CAO Accumulates More Rapidly in Cells and Have GreaterAnti-Proliferative Activity than GSAO.

4-(N—(S-cysteinylacetyl)amino)phenylarsinous acid (CAO) was produced byenzymatic cleavage of GSAO and its accumulation in endothelial cells andeffects on cell proliferation was measured.4-(N—(S-cysteinylglycylacetyl)amino)-phenylarsinous acid was produced bycleaving the γ-glutamyl group from GSAO with ovine kidney γ-glutamyltranspeptidase, and 4-(N—(S-cysteinylacetyl)amino)-phenylarsinous acid(CAO) was produced by cleaving the glycine amino acid from thisintermediate with porcine kidney aminopeptidase N (FIG. 14). The enzymeswere removed from the reactions by size-exclusion filtration.

CAO accumulated in endothelial cells at a ˜8-fold faster rate than GSAO(FIG. 16A). Cellular accumulation of these metabolites is a balancebetween rate of uptake and rate of export from the cell. GSAOaccumulation in cells is controlled by rate of export by the multidrugresistance-associated proteins (MRP) 1 and 2 (Dilda et al., 2005b). Totest whether CAO is also exported by MRP, the effect of the MRP-1inhibitor, 4H10, on accumulation in endothelial cells was measured.Cellular accumulation of CAO was increased ˜3-fold, respectively, whenMRP-1 was inhibited (FIG. 16B). This finding implies that the increasedaccumulation of CAO in endothelial cells is predominantly due toincreased rate of uptake.

The faster rate of accumulation of CAO in endothelial cells wasanticipated to result in increased anti-proliferative activity. TheIC₅₀'s for proliferation arrest of endothelial cells by GSAO and CAO in24, 48 and 72 h assays is shown in FIG. 16C. It is clear from theresults that the IC₅₀ for GSAO markedly decreases with time ofincubation and much less so for CAO. For example, the 72 h GSAO IC₅₀ issimilar to the 24 h IC₅₀ for CAO.

CAO Triggers the Mitochondrial Permeability Transition.

GSAO has been shown to inactivate the mitochondrial inner membranetransporter adenine nucleotide translocase (ANT), which leads toproliferation arrest and cell death (Don et al., 2003). CAO also inducesthe mitochondrial permeability transition (FIG. 17).

REFERENCES

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1. A compound of general formula (I):

wherein the As(OH)₂ group is para- to the N-atom on the phenyl ring; R¹is selected from hydrogen and C₁₋₃ alkyl; R² and R³ may be the same ordifferent and are independently selected from hydrogen and optionallysubstituted C₁₋₃ alkyl; R⁴ and R⁵ may be the same or different and areindependently selected from hydrogen and optionally substituted C₁₋₃alkyl; m is 1; n is 1; * indicates a chiral carbon atom; and whereineach optional substituent is independently C₁₋₃ alkyl, C₁₋₃ alkoxy,halo, hydroxyl, or hydroxy(C₁₋₃)alkyl; or a salt thereof, or anenantiomer or racemate thereof.
 2. The compound according to claim 1,wherein R¹ is selected from hydrogen, methyl and ethyl.
 3. The compoundaccording to claim 1, wherein R¹ is hydrogen.
 4. The compound accordingto claim 1, wherein R² and R³ are independently selected from hydrogen,C₁₋₃ alkyl, hydroxy(C₁₋₃)alkyl, and halo(C₁₋₃)alkyl.
 5. The compoundaccording to claim 1, wherein R² and R³ are independently selected fromhydrogen, methyl, ethyl, hydroxymethyl, and CF₃.
 6. The compoundaccording to claim 1, wherein R² and R³ are independently selected fromhydrogen, methyl and ethyl.
 7. The compound according to claim 1,wherein R² and R³ are both hydrogen.
 8. The compound according to claim1, wherein R⁴ and R⁵ are independently selected from hydrogen, C₁₋₃alkyl hydroxy-(C₁₋₃)alkyl, and halo(C₁₋₃)alkyl.
 9. The compoundaccording to claim 1, wherein R⁴ and R⁵ are independently selected fromhydrogen, methyl, ethyl, hydroxy(C₁₋₃)alkyl, and CF₃.
 10. The compoundaccording to claim 1, wherein R⁴ and R⁵ are independently selected fromhydrogen, methyl, ethyl and hydroxymethyl.
 11. The compound according toclaim 1, wherein R⁴ and R⁵ are both methyl.
 12. The compound accordingto claim 1, wherein the As(OH)₂ group is para- to the N-atom on thephenyl ring; R¹ is hydrogen or methyl; R² and R³ are independentlyselected from hydrogen, C₁₋₃ alkyl, hydroxy(C₁₋₃)alkyl andhalo(C₁₋₃)alkyl; R⁴ and R⁵ are independently selected from hydrogen,C₁₋₃ alkyl, hydroxy(C₁₋₃)alkyl and halo(C₁₋₃)alkyl; m is 1; and n is 1.13. The compound according to claim 1, wherein the As(OH)₂ group ispara- to the N-atom on the phenyl ring; R¹ is hydrogen or methyl; R² andR³ are independently selected from hydrogen, methyl, ethyl, CH₂OH, andCF₃; R⁴ and R⁵ are independently selected from hydrogen, methyl, ethyl,CH₂OH, and CF₃; m is 1; and n is
 1. 14. The compound according to claim1, wherein the As(OH)₂ group is para- to the N-atom on the phenyl ring;R¹ is hydrogen or methyl; R² and R³ are independently selected fromhydrogen, methyl and ethyl; R⁴ and R⁵ are independently selected fromhydrogen, methyl and ethyl; m is 1; and n is
 1. 15. The compoundaccording to claim 1, wherein the As(OH)₂ group is para- to the N-atomon the phenyl ring; R¹ is hydrogen or methyl; R² is hydrogen or methyl;R³ is hydrogen or methyl; R⁴ is hydrogen, methyl or ethyl; R⁵ ishydrogen or methyl; m is 1; and n is
 1. 16. The compound according toclaim 1, wherein the As(OH)₂ group is para- to the N-atom on the phenylring; R¹ is hydrogen; R² is hydrogen or methyl; R³ is hydrogen; R⁴hydrogen or methyl; R⁵ is hydrogen or methyl; m is 1; and n is
 1. 17.The compound according to claim 1 having the following structuralformula:

or a salt thereof, or an enantiomer or racemate thereof.
 18. Thecompound according to claim 17, wherein the stereochemistry at thechiral carbon denoted * is (S), and salts thereof.
 19. A pharmaceuticalcomposition comprising at least one compound of formula (I) according toclaim 1 or a salt thereof, together with a pharmaceutically acceptableexcipient, diluent or adjuvant.